Methods of treating skin conditions using inhibitors of the CD2/LFA-3 interaction

ABSTRACT

Methods of using inhibitors of the CD2/LFA-3 interaction-in treating skin conditions characterized by increased T cell activation and abnormal antigen presentation in the dermis and epidermis in mammals, including humans. Such conditions include psoriasis, UV damage, e.g., photoaging, atopic dermatitis, cutaneous T cell lymphoma such as mycosis fungoides, allergic and irritant contact dermatitis, lichen planus, alopecia areata, pyoderma gangrenosum, vitiligo, ocular cicatricial pemphigoid, and urticaria.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/730,465, filedDec. 5, 2000, which is a continuation of U.S. Ser. No. 08/466,465, filedJun. 6, 1995, now U.S. Pat. No. 6,162,432, which is acontinuation-in-part of U.S. Ser. No. 07/862,022, filed Apr. 2, 1992,now abandoned, and of PCT/US92/08755, filed Oct. 6, 1992, which is acontinuation-in-part of U.S. Ser. No. 07/770,969, filed Oct. 7, 1991,now abandoned, all of which are herein incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

This invention relates to methods of using inhibitors of the CD2/LFA-3interaction in treating skin conditions characterized by increased Tcell activation and abnormal antigen presentation in the dermis andepidermis in mammals, including humans. Such conditions includepsoriasis, UV damage, e.g., photoaging, atopic dermatitis, cutaneous Tcell lymphoma such as mycosis fungoides, allergic and irritant contactdermatitis, lichen planus, alopecia areata, pyoderma gangrenosum,vitiligo, ocular cicatricial pemphigoid, and urticaria.

BACKGROUND OF THE INVENTION

There are numerous skin conditions characterized by increased T cellactivation and abnormal antigen presentation in the dermis andepidermis. The pathophysiologic mechanisms involved in the evolution ofsuch inflammatory processes are poorly understood. However, it hasbecome apparent that skin cells are important in the generation of acutaneous inflammatory response (Kupper, “Immune and InflammatoryProcesses in Cutaneous Tissues” RELEASE, J. Clin. Invest., 86, pp.1783-89 (1990)).

The normal adult epidermal population contains 1-2% Langerhans' cellsand about 98% keratinocytes. Keratinocytes and othernonhematopoietically-derived cells resident in skin contribute to immunehomeostasis and can produce various cytokines which influence migrationof T cells and expression of adhesion molecules.

As antigen presenting cells, Langerhans' cells express a high density ofClass I[ major histocompatibility complex (MHC) antigen on the cellsurface. MHC Class II molecules bind peptides derived from endocytosedantigen and are recognized primarily by helper T lymphocytes. The T cellreceptor on T cells recognizes antigen as a peptide fragment bound tothe cell-surface molecules encoded by the MHC (Springer, “AdhesionReceptors of the Immune System”, Nature, 346, pp. 425-27 (1990)).

There are many interactions between molecules expressed on the surfaceof Langerhans' cells and the surface of T cells, in addition to the Tcell receptor/MHC interaction. These surface molecules, often referredto as adhesion molecules, participate in a number of functions includingcellular adhesion, antigen recognition, co-stimulatory signalling in Tcell activation and stimulation of effectors of T cell cytotoxicity(“Adhesion Molecules in Diagnosis and Treatment of InflammatoryDiseases”, The Lancet, 336, pp. 1351-52 (1990)). Such cell adhesionappears to be involved in activation of T cell proliferation in thegeneration of an immune response (Hughes et al., “The Endothelial Cellas a Regulator of T-cell Function”, Immunol. Rev., 117, pp. 85-102(1990)).

Various skin conditions are characterized by increased T cell activationand abnormal antigen presentation in the dermis and epidermis (Cooper,“Immunoregulation in the Skin”, in Cutaneous Lymphoma Curr. Probl.Dermatol., eds. van Vloten et al., 19, pp. 69-80 at pp. 73, 74, 76(1990)). For example, in contact allergic dermatitis, activation ofintracutaneous T cells is observed. It is known that skin from patientsexhibiting atopic dermatitis contains an increased number of Langerhans'cells (Copper, “Immunoregulation in the Skin”, in Cutaneous Lymphoma,Curr. Probl. Dermatol., eds. van Vloten et al., 19, at p. 74 (1990)). Inpsoriatic skin, there is an increased number of antigen presentingcells, composed of both Langerhans' cells and non-Langerhans' cell ClassII MHC-bearing antigen presenting cells (Cooper, “Immunoregulation inthe Skin”, in Cutaneous Lymphoma Curr. Probl. Dermatol., eds. van Vlotenet al., 19, at p. 75 (1 990)).

UV exposed skin is characterized by an overall depletion of Langerhans'cells and migration of a non-Langerhans' cell antigen-presenting cellpopulation into the epidermis, which activates autologous T cells toproliferate (Cooper, “Immunoregulation in the Skin” in CutaneousLymphoma Curr. Probl. Dermatol., eds. van Vloten et al., 19, at pp.75-76 (1990)). In human skin after 4 minimal erythemal doses of UV B,Langerhans' cells (the constitutive antigen presenting cell population)are inactivated for approximately 3 days (Cooper et al., “Effects OfUltraviolet Radiation On Human Epidermal Cell Alloantigen Presentation:Initial Depression Of Langerhans Cell-Dependent Function Is Followed ByAppearance Of T6-DR⁺ Cells That Enhance Epidermal AlloantigenPresentation”, J. Immunol., 134, pp. 129-37 (1985)). In this type of UVdamaged skin, the CD1a⁻DR⁺ macrophage population (a population ofantigen presenting cells) increases from 0% (normal skin) toapproximately 2-10% of the entire epidermal cell population and is thecell population entirely responsible for the induction of T cellproliferation to alloantigen. (Cooper et al., J. Immunol. supra (1985);Baadsgaardet al., “In Vivo Ultraviolet-Exposed Human Epidermal CellsActivate T Suppressor Cell Pathways That Involve CD4⁺ CD45RA⁺Suppressor-Inducer T Cells”, J. Immunol., 145, pp. 2854-61 (1990)).

Cutaneous T cell lymphoma is characterized by the expansion of amalignant clonal population of T cells in the dermis and epidermis.Lesional epidermal-cells contain increased numbers of CD1⁺DR⁺ antigenpresenting cells (Cooper, “Immunoregulation in the Skin” in CutaneousLymphoma, Curr. Probl. Dermatol., eds. van Vloten et al., 19, at pp.76-77 (1990)).

Presently known therapies for the above mentioned skin diseases areinadequate. Steroids or cyclosporin A are commonly used in the treatmentof psoriasis, lichen planus, urticaria, atopic dermatitis, UV damage,pyoderma gangrenosum, vitiligo, ocular cicatricial pemphigoid, alopeciaareata, allergic and irritant contact dermatitis and cutaneous T celllymphoma In addition, for some of these skin conditions, varioustherapies include retinoids, PUVA, nitrogen mustard, interferon,chemotherapy, methotrexate, UV light, antibiotics and antihistamines.See generally Fitzpatrick, Dermatology in General Medicine. 3rd ed.,McGraw Hill (1987).

Side effects to these therapies are known. Most commonly encountereddrawbacks for cyclosporin A include toxicity due to immunosuppressionand renal and neural toxicity. Steroids have well known side effectsincluding induction of Cushing Syndrome. Side effects of certain of theother aforementioned therapies include skin cancer, bone marrow andconstitutional toxicities, ligament calcification, liver fibrosis andother disorders.

T cells play a major role in the immune response by interacting withtarget and antigen presenting cells. For example, T cell-mediatedkilling of target cells is a multi-step process involving, initially,adhesion of cytolytic T cells (the effector cells) to target cells.Also, helper T cells help initiate the immune response by adhesion toantigen presenting cells.

These interactions of T cells with target and antigen presenting cellsare highly specific and depend on the recognition of an antigen on thesurface of a target or antigen presenting cell by one of the manyspecific antigen receptors on the surface of T cells.

The receptor-antigen interaction of T cells and other cells is alsofacilitated by various T cell surface proteins, e.g., theantigen-receptor complex CD3 and accessory adhesion molecules such asCD4, LFA-1, CD8, and CD2. It is also facilitated by accessory adhesionmolecules, such as LFA-3, ICAM-1 and MHC, that are expressed on thesurface of the target or antigen presenting cells. For example, LFA-1and its counter receptor ICAM-1 or ICAM-2, as well as CD2 and itscounter receptor LFA-3 have been implicated in cellular adhesion and Tcell activation. It is known that the LFA-1/ICAM and CD2/LFA-3interactions are independent.

A number of other molecules present on resting T cells have also beenimplicated in T cell adhesion, including E2 (MIC2), VLA4 (CD49d), CD44(Hermes, Pgp-1, ECMRIII) and H19 (N4) (see Makgoba et al., “TheCD2-LFA-3 and LFA-1-ICAM Pathways: Relevance to T-cell Recognition”,Immunol. Today, 10, pp. 417-22 (1989)).

One way in which T cells are activated is by binding of their antigenspecific T cell-receptors to peptide-MHC complexes on the surface ofantigen presenting cells such as macrophages. T cell activationstimulates proliferation and differentiation of two types of functionalT cells: helper cells, which promote the proliferation and maturation ofantibody-producing B lymphocytes, and killer cells, which lyse targetcells (Bierer et al., “A Monoclonal Antibody to LFA-3, the CD2 Ligand,Specifically Immobilizes Major Histocompatibility Complex Proteins”,Eur. J. Immunol., 19, pp. 661-65 (1989); Springer “Adhesion Receptors ofthe Immune System”, Nature, 346, pp. 425-34 (1990)).

The interaction between CD2 and LFA-3 remains poorly understood withrespect to activation of T cell activity. Recent studies have suggestedthat there is a specific interaction between CD2 (a T cell adhesionmolecule) and LFA-3 (a target cell and antigen presenting cell adhesionmolecule) which mediates T cell adhesion to the target or antigenpresenting cells. This cell-cell adhesion has been implicated in theinitiation of T cell functional responses (Dustin et al.,. “PurifiedLymphocyte Function Associated Antigen 3 Binds to CD2 and Mediates TLymphocyte Adhesion,” J. Exp. Med., 165, pp. 677-92 (1987); Springer etal., “The Lymphocyte Function-associated LFA-1, CD2, and LFA-3Molecules: Cell Adhesion Receptors of the Immune System”, Ann. Rev.Immunol., 5, pp. 223-52.(1987)).

LFA-3, which is found on the surface of a wide variety of cells,including human erythrocytes, has become the subject of a considerableamount of study to further elucidate its role in various T cellinteractions (see, e.g., Krensky et-al., “The Functional Significance,Distribution, and Structure of LFA-1, LFA-2, and LFA-3: Cell SurfaceAntigen Associated with CTL-Target Interactions”, J. Immunol., 131(2),pp. 611-16 (1983); Shaw et al., “Two Antigen-Independent AdhesionPathways Used by Human Cytotoxic T-cell Clones”, Nature, 323, pp. 262-64(1986)). Two natural forms of LFA-3 have been identified. One form ofLFA-3 (“transmembrane LFA-3”) is anchored in the cell membrane by atransmembrane hydrophobic domain. cDNA encoding this form of LFA-3 hasbeen cloned and sequenced (see, e.g., Wallner et al., “Primary Structureof Lymphocyte Function-Associated Antigen-3 (LFA-3)”, J. Exp. Med., 166,pp. 923-32 (1987)). Another form of LFA-3 is anchored to the cellmembrane via -a. covalent linkage to phosphatidylinositol(“PI”)-containing glycolipid. This latter form has been designated“PI-linked LFA-3”, and cDNA encoding this form of LFA-3 has also beencloned and sequenced (Wallner et al., PCT Publn. WO 90/02181).

The human CD2 (T11) molecule is a 50 kD surface glycoprotein expressedon >95% of thymocytes and virtually all peripheral T lymphocytes.Biochemical analyses using specific monoclonal antibodies have suggestedthat CD2 is T lineage-specific and exists on the cell surface in severaldifferentially glycosylated forms (Howard et al., “A Human T LymphocyteDifferentiation Marker Defined by Monoclonal Antibodies that BlockE-Rosette Formation”, J. Immunol., 126, pp. 2117-22 (1981); Brown etal., in Leukocyte Typing III, ed. McMichael, Oxford University Press,pp. 110-12 (1987); Sayre et al., “Molecular Cloning-and Expression ofT11 cDNAs Reveals a Receptor-Like Structure on Human T Lymphocytes”,Proc. Natl. Acad. Sci. USA 84. pp. 294145 (1987)).

The sequence of a human CD2 gene has been reported (Seed and Aruffo,“Molecular Cloning of the CD2 Antigen, the T-cell Erythrocyte Receptor,by a Rapid Immunoselection Procedure”, Proc. Natl. Acad. Sci. USA, 84,pp. 3365-69 (1987); Sayre et al., “Molecular Cloning and Expression ofT11 cDNAs Reveal a Receptor-like Structure on Human T Lymphocytes”,Proc. Natl. Acad. Sci. USA, 84, pp. 294145 (1987)). CD2 cDNA clonespredict a cleaved signal peptide of 24 amino acid residues, anextracellular segment of 185 residues, a transmembrane domain of 25residues and a cytoplasmic region of 117 residues (Sayre et al., supra(1987); Sewell et al., “Molecular Cloning of the Human T-LymphocyteSurface CD2 (T11) Antigen”, Proc. Natl. Acad. Sci. USA, 83, pp. 8718-22(1986); Seed and Aruffo, supra (1987); Clayton et al., Eur. J. Immunol.,17, pp. 1367-70 (1987)).

Soluble CD2 polypeptides having an LFA-3 binding domain have beenreported (PCT Publn. WO 90/08187).

Monoclonal antibodies to CD2, for example TS2/18, T11₁, T11₂, T11₃, andto LFA-3, for example TS2/9, have also been reported (see, e.g., Hugheset al., “The Endothelial Cell as a Regulator of T-Cell Function”,Immunol. Reviews, 117, pp. 85-102 (1990); Meuer, “An Alternative Pathwayof T-Cell Activation: A Functional Role for the 50 kD T11 SheepErythrocyte Receptor Protein”, Cell, 36, pp. 897-906 (1984)).

The need still exists for improved methods of preventing and treatingskin conditions exhibiting increased T cell activation and abnormalantigen presentation.

SUMMARY OF THE INVENTION

The present invention generally solves many of the problems referred toabove. It for the first time provides a method of preventing or treatingskin conditions, characterized by increased T cell activation andabnormal antigen presentation in the dermis and epidermis, in a mammal,whereby an inhibitor of the CD2/LFA-3 interaction, is administered tothe mammal. The methods of this invention are superior to previouslyavailable therapies for these skin conditions for many reasons,including less immunosuppression than pre-existing therapies and morespecific therapy with less general toxicity.

The method of the present invention preferably will be used in thetreatment or prophylaxis of skin conditions selected from psoriasis, UVdamage, e.g., photoaging, atopic dermatitis, cutaneous T cell lymphomasuch as mycosis fungoides, allergic and irritant contact dermatitis,lichen planus, alopecia areata, pyoderma gangrenosum, vitiligo, ocularcicatricial pemphigoid, and urticaria, preferably psoriasis or UVdamage.

Inhibitors that can be used in accordance with the method of the presentinvention include any molecule that inhibits the CD2/LFA-3 interaction.Preferably, the inhibitor is selected from the group consisting ofanti-LFA-3 antibody homologs, anti-CD2 antibody homologs soluble LFA-3polypeptides, small molecules, e.g., carbohydrates, soluble CD2polypeptides, CD2 or LFA-3 mimetic agents and derivatives thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the percent inhibition caused by an anti-LFA-3monoclonal antibody (7A6) or an anti-CD2 monoclonal antibody (TS2/18) ascompared to a non-specific control IgG₁ antibody (MOPC21) of autologousT cell activation by psoriatic epidermal cells in 4 patients.

FIG. 2 illustrates the inhibition of allogeneic-T cell activation by UVdamaged epidermal cells ([³H]TdR incorporation) caused by an anti-LFA-3monoclonal antibody (1E6) or an anti-CD2 monoclonal antibody (TS2/18) ascompared to a non-specific IgG antibody (MOPC21).

FIG. 3 illustrates the inhibition of autologous mononuclear cellresponses to psoriatic epidermal cells ([³H]TdR incorporation) caused byan LFA3TIP fusion molecule as compared to a human IgG control.

FIG. 4 illustrates the effect of LFA3TIP on spontaneous lesionalpsoriatic dermal cell proliferation. A reduction in spontaneous dermalcell proliferation ([³H]TdR incorporation) was seen with dose responsesbetween 0.03 and 0.003 μg/ml of LFA3TIP. LFA3TIP cultures areconsistently less proliferative than the human IgG control culturesthrough the concentration of 0.01 μg/ml.

FIG. 5 illustrates the inhibition of allogeneic mononuclear cellresponses to normal epidermal cells ([³H]TdR incorporation) caused by anLFA3TIP fusion molecule as compared to a human IgG control.

FIG. 6 illustrates the inhibition of autologous mononuclear cellresponses to psoriatic dermal cells ([³H]TdR incorporation) caused by anLFA3TIP fusion molecule as compared to a human IgG control.

FIG. 7 illustrates the inhibition of inflammatory macrophage APCactivity in UV-exposed epidermal cells (EC) ([³H]TdR incorporation)caused by an LFA3TIP fusion molecule as compared to a human IgG control.Two concentrations of UV-EC are shown in panel 7A and 7B. LFA3TIPincubation resulted in approximately 50% inhibition.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, “CD2” means a CD2 polypeptide that binds to a naturallyoccurring LFA-3 polypeptide and which is encoded by (a) a naturallyoccurring mammalian CD2 DNA sequence (e.g., SEQ ID NO:5); (b) a DNAsequence degenerate to a naturally occurring CD2 DNA sequence; or (c) aDNA sequence that hybridizes to one of the foregoing DNA sequences underconditions equivalent to about 20° C. to 27° C. below T_(m) and 1 Msodium chloride.

As used herein, “LFA-3” means an LFA-3 polypeptide that binds to anaturally occurring CD2 polypeptide and which is encoded by (a) anaturally occurring mammalian LFA-3 DNA sequence (e.g., SEQ ID NO:1 orSEQ ID NO:3); (b) a DNA sequence degenerate to a naturally occurringLFA-3 DNA sequence; or (c) a DNA sequence that hybridizes to one of theforegoing DNA sequences under conditions to about 20° C. to 27° C. belowT_(m) and 1 M sodium chloride.

As used herein, a “soluble LFA-3 polypeptide” or a “soluble CD2polypeptide” is an LFA-3 or CD2 polypeptide incapable of anchoringitself in a membrane. Such soluble polypeptides include, for example,CD2 and LFA-3 polypeptides that lack a sufficient portion of theirmembrane spanning domain to anchor the polypeptide or are modified suchthat the membrane spanning domain is non-functional. As used hereinsoluble LFA-3 polypeptides include full-length or truncated (e.g., withinternal deletions) PI-linked LFA-3.

As used herein, an “antibody homolog” is a protein comprising-one ormore polypeptides selected from immunoglobulin light chains,immunoglobulin heavy chains and antigen-binding fragments thereof whichare capable of binding to one or more antigens. The componentpolypeptides of an antibody homolog composed of more than onepolypeptide may optionally be disulfide-bound or otherwise covalentlycrosslinked. Accordingly, antibody homologs include intactimmunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypesthereof), wherein the light chains of the immunoglobulin may be of typeskappa or lambda. Antibody homologs also include portions of intactimmunoglobulins that retain antigen-binding specificity, for example,Fab fragments, Fab′ fragments, F(ab′)2 fragments, F(v) fragments, heavychain monomers or dimers, light chain monomers or dimers, dimersconsisting of one heavy and one light chain, and the like.

As used herein, a “humanized recombinant antibody homolog” is anantibody homolog, produced by recombinant DNA technology, in which someor all of the amino acids of a human immunoglobulin light or heavy chainthat are not required for antigen binding have been substituted for thecorresponding amino acids from a nonhuman mammalian immunoglobulin lightor heavy chain.

As used herein, a “chimeric recombinant antibody homolog” is an antibodyhomolog, produced by recombinant DNA technology, in which all or part ofthe hinge and constant regions of an immunoglobulin light chain, heavychain, or both, have been substituted for the corresponding regions fromanother immunoglobulin light chain or heavy chain.

Skin Conditions

The methods of this invention are useful to prevent or treat mammalian,including human, skin conditions characterized by increased T cellactivation and abnormal antigen presentation in the dermis andepidermis, by administering inhibitors of the CD2/LFA-3 interaction.Such conditions include psoriasis, UV damage, atopic dermatitis,cutaneous T cell lymphoma such as mycosis fungoides, allergic andirritant contact dermatitis, lichen planus, alopecia areata, pyodermagangrenosum, vitiligo, ocular cicatricial pemphigoid, and urticaria. Itis to be understood that methods of treatment and prophylaxis of skinconditions such as pyoderma gangrenosum and urticaria are includedwithin the scope of the present invention. These latter skin conditionsare also cyclosporin A sensitive dermatoses and therefore involve T cellactivation. Preferably, the methods of the invention are used in theprophylaxis or treatment of psoriasis or UV damage. The methods of theinvention may be practiced on any mammal, preferably on humans.

While not wishing to be bound by theory, applicants believe thatinhibitors of the CD2/LFA-3 interaction used in accordance with themethods of this invention are prophylactic and therapeutic for thetreatment of the aforementioned skin conditions because they inhibit theinteraction between T cells and antigen presenting cells, resulting in,among other things, an inhibition of T cell proliferation andactivation. Applicants believe that adverse effects of skin conditionsof the type discussed herein are due to such T cell proliferation andactivation. Applicants believe that the methods of the present inventionare superior to previously available therapies for these skin conditionsfor a number of reasons, including, inhibition of antigen 5specificinteractions for all antigens present, inhibition of T cell activation,no general immunosuppression and, possibly, induction of tolerance.

In particular, applicants believe that use of the methods of thisinvention will result in more specific targeting of therapy to T cellsactually in the initiating stage of the lesion with no effect onpolymorphonuclear leukocytes or macrophage mediated effector mechanisms.Accordingly, the patient will be less susceptible to infections thanwith steroids or other general immunosuppressants. Thus, methods ofinhibiting T cell activation, as provided herein, are prophylactic andtherapeutic for such skin conditions.

Inhibitors of the CD2/LFA-3 Interaction

Any inhibitor of the CD2/LFA-3 interaction is useful in the methods ofthis invention.

Such inhibitors include anti-LFA-3 antibody homologs, anti-CD2 antibodyhomologs, soluble LFA-3 polypeptides, soluble CD2 polypeptides, smallmolecules, e.g., carbohydrates, LFA-3 and CD2 mimetic agents andderivatives thereof Preferred inhibitors are soluble LFA-3 polypeptidesand anti-LFA-3 antibody homologs.

The utility in the methods of this invention of specific soluble CD2polypeptides, soluble LFA-3 polypeptides, anti-LFA-3 antibody homologs,anti-CD2 antibody homologs or CD2 and LFA-3 mimetic agents may easily bedetermined by assaying their ability to inhibit the LFA-3/CD2interaction. This ability may be assayed, for example, using a simplecell binding assay that permits visual (under magnification) evaluationof the ability of the putative inhibitor to inhibit the interactionbetween LFA-3 and CD2 on cells bearing these molecules. Jurkat cells arepreferred as the CD2⁺ substrate and sheep red blood cells or human JYcells are preferred as the LFA-3⁺ substrate. The binding characteristicsof soluble polypeptides, antibody homologs and mimetic agents useful inthis invention may be assayed in several known ways, such as byradiolabeling the antibody homolog, polypeptide or agent (e.g., ³⁵S or¹²⁵I) and then contacting the labeled polypeptide, mimetic agent-orantibody homolog with CD2⁺ of LFA-3⁺ cells, as appropriate. Bindingcharacteristics may also be assayed using an appropriate enzymaticallylabelled secondary antibody. Rosetting competition assays such as thosedescribed by Seed et al. (Proc. Natl. Acad. Sci. USA, 84, pp. 3365-69(1987)) may also be used.

A. Anti-LFA-3 and Anti-CD2 Antibody Homologs

Many types of anti-LFA-3 or anti-CD2 antibody homologs are useful in themethods of this invention. These include monoclonal antibodies,recombinant antibodies, chimeric recombinant antibodies, humanizedrecombinant antibodies, as well as antigen-binding portions of theforegoing.

Among the anti-LFA-3 antibody homologs, it is preferable to usemonoclonal anti-LFA-3 antibodies. It is more preferable to use amonoclonal anti-LFA-3 antibody produced by a hybridoma selected from thegroup of hybridomas having Accession Nos. ATCC HB 10693 (1E6), ATCC HB10694 (HC-1B11), ATCC HB 10695 (7A6), and ATCC HB 10696 (8B8), or themonoclonal antibody known as TS2/9 (Sanchez-Madrid et al., “ThreeDistinct Antigens Associated with Human T-Lymphocyte-Mediated Cytolysis:LFA-1, LFA-2 and LFA-3”, Proc. Natl. Acad. Sci. USA 79, pp. 7489-9³(1982)). Most preferably, the monoclonal anti-LFA-3 antibody is producedby a hybridoma selected from the group of hybridomas having AccessionNos. ATCC HB 10695 (7A6) and ATCC HB 10693 (1E6).

Among the anti-CD2 antibody homologs, it is preferable to use monoclonalanti-CD2 antibodies, such as the anti-CD2 monoclonal antibodies known asthe T11₁ epitope antibodies, including TS2/18 (Sanchez-Madrid et al.,“Three Distinct Antigens Associated with Human T-Lymphocyte-MediatedCytolysis: LFA-1, LFA-2 and LFA-3”, Proc. Natl. Acad. Sci. USA. 79, pp.7489-93 (1982)).

The technology for producing monoclonal antibodies is well known.Briefly, an immortal cell line (typically myeloma cells) is fused tolymphocytes (typically splenocytes) from a mammal immunized withpreparation comprising a given antigen, and the culture supernatants ofthe resulting hybridoma cells are screened for antibodies against theantigen. See generally, Kohler et al., Nature, “Continuous Cultures ofFused Cells Secreting Antibody of Predefined Specificity”, 256, pp.495-97 (1975). Useful immunogens for the purpose of this inventioninclude CD2- or LFA-3-bearing cells, as well as cell free preparationscontaining LFA-3, CD2 or counter receptor-binding fragments thereof(e.g., CD2 fragments that bind to LFA-3 or LFA-3 fragments that bind toCD2).

Immunization may be accomplished using standard procedures. The unitdose and immunization regimen depend on the species of mammal immunized,its immune status, the body weight of the mammal, etc. Typically, theimmunized mammals are bled and the serum from each blood sample isassayed for particular antibodies using appropriate screening assays.For example, useful anti-LFA-3 or anti-CD2 antibodies may be identifiedby testing the ability of the immune serum to block sheep-red blood cellrosetting of Jurkat cells, which results from the presence of LFA-3 andCD2 on the respective surfaces of these cells. The lymphocytes used inthe production of hybridoma cells typically are isolated from immunizedmammals whose sera have already tested positive for the presence of thedesired antibodies using such screening assays.

Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. Preferred immortalcell lines are mouse myeloma cell lines that are sensitive to culturemedium containing hypoxanthine, aminopterin and thymidine (“HATmedium”).

Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”) 3350. Hybridoma cellsresulting from the fusion are then selected using HAT medium, whichkills unfused and unproductively fused myeloma cells (unfusedsplenocytes die after several days because they are not transformed).Hybridomas producing a desired antibody are detected by screening thehybridoma culture supernatants, for example, for the ability to bind totheir respective counter receptor, or for their ability to block Jurkatcell adhesion to sheep red blood cells. Subcloning of the hybridomacultures by limiting dilution is typically performed to ensuremonoclonality.

To produce anti-LFA-3 or anti-CD2 monoclonal antibodies, hybridoma cellsthat tested positive in such screening assays are cultured in a nutrientmedium under conditions and for a time sufficient to allow the hybridomacells to secrete the monoclonal antibodies into the culture medium.Tissue culture techniques and culture media suitable for hybridoma cellsare well known. The conditioned hybridoma culture supernatant may becollected and the desired antibodies optionally further purified bywell-known methods.

Alternatively, the desired antibody may be produced by injecting thehybridoma cels into the peritoneal cavity of a pristane-primed mouse.The hybridoma cells proliferate in the peritoneal cavity, secreting theantibody, which accumulates as ascites fluid. The antibody may beharvested by withdrawing the ascites fluid from the peritoneal cavitywith a syringe.

Anti-CD2 and anti-LFA-3 antibody homologs useful in the presentinvention may also be recombinant antibodies produced by host cellstransformed with DNA encoding immunoglobulin light and heavy chains of adesired antibody. Recombinant antibodies may be produced by well knowngenetic engineering techniques. See, e.g., U.S. Pat. No. 4,816,397,which is incorporated herein by reference.

For example, recombinant antibodies may be produced by cloning cDNA orgenomic DNA encoding the immunoglobulin light and heavy chains of thedesired antibody from a hybridoma cell that produces an antibody homologuseful-in this invention. The cDNA or genomic DNA encoding thosepolypeptides is then inserted into expression vectors so-that both genesare operatively linked to their own transcriptional and translationalexpression control sequences. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. Typically, both genes are inserted into the same expressionvector.

Prokaryotic or eukaryotic host cells may be used. Expression ineukaryotic host cells is preferred because such cells are more likelythan prokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody. However, any antibody produced that isinactive due to improper folding may be renaturable according to wellknown methods (Kim and Baldwin, “Specific Intermediates in the FoldingReactions of Small Proteins and the Mechanism of Protein Folding”, Ann.Rev. Biochem., 51, pp. 459-89 (1982)). It is possible that the hostcells will produce portions of intact antibodies, such as light chaindimers or heavy chain dimers, which also are antibody homologs accordingto the present invention.

It will be understood that variations on the above procedure are usefulin the present invention. For example, it may be desired to transform ahost cell with DNA encoding either the light chain or the heavy chain(but not both) of an antibody homolog. Recombinant DNA technology mayalso be used to remove some or all of the DNA encoding either or both ofthe light and heavy chains that is not necessary for CD2 or LFA-3counter receptor binding. The molecules expressed from such truncatedDNA molecules are useful in the methods of this invention. In addition,bifunctional antibodies may be produced in which one heavy and one lightchain are anti-CD2 or anti-LFA-3 antibody homologs and the other heavyand light chain are specific for an antigen other than CD2 or LFA-3, oranother epitope of CD2 or LFA-3.

Chimeric recombinant anti-LFA-3 or anti-CD2 antibody homologs may beproduced by transforming a host cell with a suitable expression vectorcomprising DNA encoding the desired immunoglobulin light and heavychains in which all or some of the DNA encoding the hinge and constantregions of the heavy and/or the light chain have been substituted withDNA from the corresponding region of an immunoglobulin light or heavychain of a different species. When the original recombinant antibody isnonhuman, and the inhibitor is to be administered to a human,substitution of corresponding human sequences is preferred. An exemplarychimeric recombinant antibody has mouse variable regions and human hingeand constant regions. See generally, U.S. Pat. No. 4,816,397 andMorrison et al., “Chimeric Human Antibody Molecules: MouseAntigen-Binding Domains With Human Constant Region Domains”, Proc. Natl.Acad. Sci. USA, 81, pp. 6851-55 (1984).

Humanized recombinant anti-LFA-3 or anti-CD2 antibodies may be producedby transforming a host cell with a suitable expression vector comprisingDNA encoding the desired nonhuman immunoglobulin light and heavy chainsin which all or some of the DNA encoding amino acids not involved inantigen binding have been substituted with DNA from the correspondingregion of a desired human immunoglobulin light or heavy chain. Seegenerally, Jones et al., “Replacing the Complementary-DeterminingRegions in a Human Antibody with Those from a Mouse”, Nature. 321, pp.522-25 (1986).

Anti-CD2 and anti-LFA-3 antibody homologs that are not intact antibodiesare also useful in this invention. Such homologs may be derived from anyof the antibody homologs described above. For example, antigen-bindingfragments, as well as full-length monomeric, dimeric or trimericpolypeptides derived from the above-described antibodies are themselvesuseful. Useful antibody homologs of this type include Fab fragments,Fab′ fragments, F(ab′)₂ fragments, F(v) fragments, heavy chain monomersor dimers, light chain monomers or dimers, dimers consisting of oneheavy and one light chain, and the like. Anti-LFA-3 heavy chains arepreferred anti-LFA-3 antibody fragments.

Antibody fragments may also be produced by chemical methods, e.g., bycleaving an intact antibody with a protease, such as pepsin or papain,and optionally treating the cleaved product with a reducing-agent.Alternatively, useful fragments may be produced by using host cellstransformed with truncated heavy and/or light chain genes. Heavy andlight chain monomers may be produced by treating an intact antibody witha reducing agent, such as dithiothreitol, followed by purification toseparate the chains. Heavy and light chain monomers may also be producedby host cells transformed with DNA encoding either the desired heavychain or light chain, but not both. See,.e.g., Ward et al., “BindingActivities of a Repertoire of Single Immunoglobulin Variable DomainsSecreted from Escherichia coli”, Nature, 341, pp. 544-46 (1989); Sastryet al., “Cloning of the Immunological Repertoire in Escherichia coli forGeneration of Monoclonal Catalytic Antibodies: Construction of a HeavyChain Variable Region-Specific cDNA Library”. Proc. Natl. Acad. Sci.USA, 86, pp. 5728-32 (1989).

B. Soluble CD2 and LFA-3 Polypeptides

Soluble LFA-3 polypeptides or soluble CD2 polypeptides that inhibit theinteraction of LFA-3 and CD2 are useful in the methods of the presentinvention. Soluble LFA-3 polypeptides are preferred.

Soluble LFA-3 polypeptides may be derived from the transmembrane form ofLFA-3, particularly the extracellular domain (e.g., AA₁-AA₁₈₇ of SEQ IDNO:2). Such polypeptides are described in U.S. Pat. No. 4,956,281 andco-pending U.S. patent application Ser. No. 07/667,971 (which shares acommon assignee with the present application), which are hereinincorporated by reference. Preferred soluble LFA-3 polypeptides includepolypeptides consisting of AA₁-AA₉₂ of SEQ ID NO:2, AA₁-AA₈₀ of SEQ IDNO:2i AA₅₀-AA₆₅ of SEQ ID NO:2 and AA₂₀-AA₈₀ of SEQ ID NO:2. A vectorcomprising a DNA sequence encoding SEQ ID. NO:2 (i.e., SEQ ID NO:1) isdeposited with the American Type Culture Collection under Accession No.75107.

The most preferred fusion proteins of this type contain the aminoterminal 92 amino acids of mature LFA-3, the C-terminal 10 amino acidsof a human IgG1 hinge region containing the two cysteine residuesthought to participate in interchain disulfide bonding, and the C_(H)2and C_(H)3 regions of a human IgG1 heavy chain constant domain (e.g.,SEQ ID NO:8). This fusion protein is referred to herein as “LFA3TIP.” Aplasmid, pSAB152, encoding an exemplary LFA3TIP is deposited withAmerican Type Culture Collection under the accession number ATCC 68720.The DNA sequence of the pSAB152 insert is SEQ ID NO:7.

One way of producing LFA3TIP for use in the methods of this invention isdescribed in co-pending, commonly assigned U.S. patent application Ser.No. 07/770,967. Generally, conditioned culture medium of COS7 or CHOcells transfected with pSAB 152 was concentrated using an AMICON® S1Y30spiral cartridge system (AMICON®, Danvers, Mass.) and subjected toProtein A-Sepharose 4B® (Sigma, St. Louis, Mo.) chromatography. Thebound proteins were eluted and subjected to Superose-12 (Pharmacia/LKB,Piscataway, N.J.) gel filtration chromatography.

Superose-12 fractions containing LFA3TIP with the least amount ofcontaminating proteins, as determined on SDS-PAGE gels and by Westernblot analysis, (see, e.g., Towbin et al., Proc. Natl. Acad. Sci. USA.74, pp. 4350-54 (1979); Antibodies: A Laboratory Manual, pp. 474-510(Cold Spring Harbor Laboratory (1988)), were pooled and concentrated ina YM30 Centricon (AMCON®). LFA3TIP was detected on Western blots using arabbit anti-LFA-3 polyclonal antiserum, followed by detectably labeledgoat anti-rabbit IgG. The purified LFA3TIP of COS7 or CHO cells was adimer of two monomeric LFA-3-Ig fusion proteins, connected by disulfidebonds.

Another preferred fusion protein consists of the first and second LFA-3domain fused to the hinge C_(H) ² and C_(H) ³ regions of human IgG1,herein referred to as LLFA3-Ig.

Soluble LFA-3 polypeptides may also be derived from the PI-linked formof LFA-3, such as those described in PCT Patent Application Serial No.WO 90/02181. A vector comprising a DNA sequence encoding PI-linked LFA-3(i.e., SEQ ID NO:3) is deposited with the American Type CultureCollection under Accession No. 68788. It is to be understood thatthe-PI-linked form of LFA-3 and the transmembrane form of LFA-3 haveidentical amino acid sequences through the entire extracellular domain.Accordingly, the preferred PI-linked LFA-3 polypeptides are the same asfor the transmembrane form of LFA-3.

Soluble CD2 polypeptides may be derived from full length CD2,particularly the extracellular domain (e.g., AA₁-AA₁₈₅ of SEQ ID NO:6).Such polypeptides may comprise all or part of the extracellular domainof CD2. Exemplary soluble CD2 polypeptides are described in PCT WO90/08187, which is herein incorporated by reference.

The production of the soluble polypeptides useful in this invention maybe achieved by a variety of methods known in the art. For example, thepolypeptides may be derived from intact transmembrane LFA-3 or CD2molecules or an intact PI-linked LFA-3 molecule by proteolysis usingspecific endopeptidases in combination with exopeptidases, Edmandegradation, or both. The intact LFA-3 molecule or the intact CD2molecule, in turn, may be purified from its natural source usingconventional methods. Alternatively, the intact LFA-3 or CD2 may beproduced by known recombinant DNA techniques using cDNAs (see, e.g.,U.S. Pat. No. 4,956,281 to Wallner et al.; Aruffo and Seed, Proc. Natl.Acad. Sci., 84, pp. 2941-45 (1987); Sayre et al., Proc. Natl. Acad. Sci.USA, 84, pp. 2941-45 (1987)).

Preferably, the soluble polypeptides useful in the present invention areproduced directly, thus eliminating the need for an entire LFA-3molecule or an entire CD2 molecule as a starting material. This may beachieved by conventional chemical synthesis techniques or by well-knownrecombinant DNA techniques wherein only those DNA sequences which encodethe desired peptides are expressed in transformed hosts. For example, agene which encodes the desired soluble LFA-3 polypeptide or soluble CD2polypeptide may be synthesized by chemical means using anoligonucleotide synthesizer. Such oligonucleotides are designed based onthe amino acid sequence of the desired soluble LFA-3 polypeptide orsoluble CD2 polypeptide. Specific DNA sequences coding for the desiredpeptide also can be derived from the full length DNA sequence byisolation of specific restriction endonuclease fragments or by PCRsynthesis of the specified region.

Standard methods may be applied to synthesize a gene encoding a solubleLFA-3 polypeptide or a soluble CD2 polypeptide that is useful in thisinvention. For example, the complete amino acid sequence may be used toconstruct a back-translated gene. A DNA oligomer containing a nucleotidesequence coding for a soluble LFA-3 polypeptide or a soluble CD2polypeptide useful in this invention may be synthesized in a singlestep. Alternatively, several smaller oligonucleotides coding forportions of the desired polypeptide may be synthesized and then ligated.Preferably, a soluble LFA-3 polypeptide or a soluble CD2 polypeptideuseful in this invention will be synthesized as several separateoligonucleotides which are subsequently linked together. The individualoligonucleotides typically contain 5′ or 3′ overhangs for complementaryassembly.

Once assembled, preferred genes will be characterized by sequences thatare recognized by restriction endonucleases (including uniquerestriction sites for direct assembly into a cloning or an expressionvector), preferred codons taking into consideration the host expressionsystem to be used, and a sequence which, when transcribed, produces astable, efficiently translated mRNA. Proper assembly may be confirmed bynucleotide sequencing, restriction mapping, and expression of abiologically active polypeptide in a suitable host.

It will be appreciated by those of skill in the art that, due to thedegeneracy of the genetic code, DNA molecules comprising many othernucleotide sequences will also be capable of encoding the soluble LFA-3and CD2 polypeptides encoded by the specific DNA sequences describedabove. These degenerate sequences also code for polypeptides that areuseful in this invention.

The DNA sequences may be expressed in unicellular hosts. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host. Preferably, the expressioncontrol sequences, and the gene of interest, will be contained in anexpression vector that further comprises a-bacterial selection markerand origin of replication. If the expression host is a eukaryotic cell,the expression vector should further comprise an additional expressionmarker useful in the expression host.

The DNA sequences encoding the desired soluble polypeptides may or maynot encode a signal sequence. If the expression host is prokaryotic, itgenerally is preferred that the DNA sequence not encode a signalsequence. If the expression host is eukaryotic, it generally ispreferred that a signal sequence be encoded.

An amino terminal methionine may or may not be present on the expressedproduct. If the terminal methionine is not cleaved by the expressionhost, it may, if desired, be chemically removed by standard techniques.

A wide variety of expression host/vector combinations may be employed.Useful expression vectors for eukaryotic hosts, include, for example,vectors comprising expression control sequences from SV40, bovinepapilloma virus, adenovirus and cytomegalovirus. Useful expressionvectors for bacterial hosts include known bacterial plasmid, such asplasmids from E. coli, including col E1, pCR1, pBR322,.pMB9 and theirderivatives, wider host range plasmids, such as RP4, phage DNAs, e.g.,the numerous derivatives of phage lambda, e.g., NM989, and other DNAphages, such as M13 and filamentous single stranded DNA phages. Usefulexpression vectors for yeast cells include the 2p plasmid andderivatives thereof. Useful vectors for insect cells include pVL 941.

In addition, any of a wide variety of expression control -sequences maybe used in these vectors. Such useful expression control sequencesinclude the expression control sequences associated with structuralgenes of the foregoing expression vectors. Examples of useful expressioncontrol sequences include, for example, the early and late promoters ofSV40 or adenovirus, the lac system, the to system, the TAC or TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3′-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating system and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof.

A wide variety of unicellular host cells are useful. These hosts mayinclude well known eukaryotic and prokaryotic hosts, such as strains ofE. coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cellssuch as Spodontera frugiredrda (SF9), animal cells such as CHO and mousecells, African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40,and BMT 10, and human cells, as well as plant cells in tissue culture.For animal cell expression, we prefer CHO cells and COS 7 cells.

It should, of course, be understood that not all vectors and expressioncontrol-sequences will function equally well to express the DNAsequences described herein. Neither will all hosts function equally wellwith the same expression system. However, one of skill in the art maymake a selection among these vectors, expression control sequences andhosts without undue experimentation. For example, in selecting a vector,the host must be considered because the vector must replicate in it. Thevector's copy number, the ability to control that copy number, and theexpression of any other proteins encoded by the vector, such asantibiotic markers, should also be considered.

In selecting an expression control sequence, a variety of factors shouldalso be considered. These include, for example, the relative strength ofthe sequence, its controllability, and its compatibility with the DNAsequences discussed herein, particularly as regards potential secondarystructures. Unicellular hosts should be selected by consideration oftheir compatibility with the chosen vector, the toxicity of the productcoded for by the DNA sequences, their secretion characteristics, theirability to fold the soluble polypeptides correctly, their fermentationor culture requirements, and the ease of purification of the productscoded for by the DNA sequences.

Within these parameters, one of skill in the art may select variousvector/expression control sequence/host combinations that will expressthe desired DNA sequences on fermentation or in large scale animalculture, for example with CHO cells or COS 7 cells.

The soluble LFA-3 and CD2 polypeptides may be isolated from thefermentation or cell culture and purified using any of a variety ofconventional methods. One of skill in the art may select the mostappropriate isolation and purification techniques.

While recombinant DNA techniques are the preferred method of producinguseful soluble CD2 polypeptides or soluble LFA-3 polypeptides having asequence of more than 20 amino acids, shorter CD2 or LFA-3 polypeptideshaving less than about 20 amino acids are preferably produced byconventional chemical synthesis techniques. Synthetically producedpolypeptides useful in this invention can advantageously be produced inextremely high yields and can be easily purified.

Preferably, such soluble CD2 polypeptides or soluble LFA-3 polypeptidesare synthesized by solution phase or solid phase polypeptide synthesisand, optionally, digested with carboxypeptidase (to remove C-terminalamino acids) or degraded by manual Edman degradation (to removeN-terminal amino acids). Proper folding of the polypeptides may beachieved under oxidative conditions which favor disulfide bridgeformation as described by Kent, “Chemical Synthesis of Polypeptides andProteins”, Ann. Rev. Biochem., 57, pp. 957-89 (1988). Polypeptidesproduced in this way may then be purified by separation techniqueswidely known in the art, preferably utilizing reverse phase HPLC. Theuse of solution phase synthesis advantageously allows for the directaddition of certain derivatized amino acids to the growing polypeptidechain, such as the O-sulfate ester of tyrosine. This obviates the needfor a subsequent derivatization step to modify any residue of thepolypeptides useful in this invention.

C. LFA-3 and CD2 Mimetic Agents

Also useful in the methods of this invention are LFA-3 and CD2 mimeticagents. These agents which may be peptides, semi-peptidic compounds ornon-peptidic compounds, are inhibitors of the CD2/LFA-3 interaction. Themost preferred CD2 and LFA-3 mimetic agents will inhibit the CD2/LFA-3interaction at least as well as anti-LFA-3 monoclonal antibody 7A6 oranti-CD2 monoclonal antibody TS2/18 (described supra).

Such mimetic agents may be produced by synthesizing a plurality ofpeptides (e.g., 5-20 amino acids in length), semi-peptidic compounds ornon-peptidic, organic compounds, and then screening those compounds-fortheir ability to inhibit the CD2/LFA-3 interaction. See generally U.S.Pat. No. 4,833,092, Scott-and-Smith, “Searching for Peptide Ligands withan Epitope Library”, Science 249, pp.386-90 (1990), and Devlin et al.,“Random Peptide Libraries: A Source of Specific Protein BindingMolecules”, Science, 249, pp. 404-07 (1990), which are hereinincorporated by reference.

D. Derivatized Inhibitors

Also useful in the methods of this invention are derivatized inhibitorsof the CD2/LFA-3 interaction in which, for example, any of the antibodyhomologs, soluble CD2 and LFA-3 polypeptides, or CD2 and LFA-3 mimeticagents described herein are functionally linked (by chemical coupling,genetic fusion or otherwise) to one or more members independentlyselected from the group consisting of anti-LFA-3 and anti-CD2 antibodyhomologs, soluble LFA-3 and CD2 polypeptides, CD2 and LFA-3 mimeticagents, cytotoxic agents and pharmaceutical agents.

One type of derivatized inhibitor is produced by crosslinking two ormore inhibitors (of the same type or of different types). Suitablecrosslinkers include those that are heterobifunctional, having twodistinctly reactive groups separated by an appropriate spacer (e.g.,m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional(e.g., disuccininmidyl suberate). Such linkers are available from PierceChemical Company, Rockford, Ill.

Another possibility for cross-linking takes advantage of the PI linkagesignal sequence in PI-linked LFA-3, or fragments thereof. Specifically,DNA encoding the PI-linkage signal sequence (e.g., AA₁₆₂-AA₂₁₂ of SEQ IDNO:4) is ligated downstream of DNA encoding a desired polypeptide,preferably a soluble LFA-3 polypeptide. If this construct is expressedin an appropriate eukaryotic cell, the cell will recognize the PIlinkage signal sequence and will covalently link PI to the polypeptide.The hydrophobic property of the PI may then be exploited to formmicellar aggregates of the polypeptides.

Also useful are inhibitors linked to one or more cytotoxic orpharmaceutical agents. Useful pharmaceutical agents include biologicallyactive peptides, polypeptides and proteins, such as antibody homologsspecific for a human polypeptide other than CD2 or LFA-3, or portionsthereof. Useful pharmaceutical agents and cytotoxic agents also includecyclosporin A, prednisone, FK506, methotrexate, steroids, retinoids,interferon, and nitrogen mustard.

Preferred inhibitors derivatized with a pharmaceutical agent includerecombinantly-produced polypeptides in which a soluble LEA-3polypeptide, soluble CD2 polypeptide, or a peptidyl CD2 or peptidylLFA-3 mimetic agent is fused to all or part of an immunoglobulin heavychain hinge region and all or part of a heavy chain constant region.Preferred polypeptides for preparing such fusion proteins are solubleLFA-3 polypeptides. Most preferred are fusion proteins containingAA₁-AA₉₂ of LFA-3 (e.g., SEQ ID NO:2) fused to a portion of a human IgG1hinge region (including the C-terminal ten amino acids of the hingeregion containing two cysteine residues thought to participate ininterchain disulfide bonding) and the C_(H)2 and C_(H)3 regions of anIgG1 heavy chain constant domain. Such fusion proteins are expected toexhibit prolonged serum half-lives and enable inhibitor dimerization.

Pharmaceutical Compositions and Methods According to this Invention

This invention provides a method for preventing or treating theabove-mentioned skin conditions in a mammal by administering to themammal one or more inhibitors of the CD2/LFA-3 interaction, orderivatized form(s) thereof.

Preferably, an effective amount of the inhibitor or derivatized formthereof is administered. By “effective amount” is meant an amountcapable of lessening the spread or severity of the skin conditionsdescribed herein.

It will be apparent to those of skill in the art that the effectiveamount of inhibitor will depend, inter alia, upon the administrationschedule, the unit dose administered, whether the inhibitor isadministered in combination with other therapeutic agents, the immunestatus and health of the patient, the therapeutic or prophylacticactivity of the particular inhibitor administered and the serumhalf-life.

Preferably, the inhibitor is administered at a dose between about 0.001and about,50 mg inhibitor per kg body weight, more preferably, betweenabout 0.01 and about 10 mg inhibitor per kg body weight, most preferablybetween about 0.1 and about 4 mg inhibitor per kg body weight.

Unit doses should be administered until an effect is observed. Theeffect may be measured by a variety of methods, including, in vitro Tcell activity assays and clearing of affected skin areas. Preferably,the unit dose is administered about one to three times per week or oneto three times per day. More preferably, it is administered about one tothree times per day for between about 3 and 7 days, or about one tothree times per day for between about 3 and 7 days on a monthly basis.It will be recognized, however, that lower or higher dosages and otheradministrations schedules may be employed.

The inhibitor(s) or derivatized form(s) thereof are also preferablyadministered in a composition including a pharmaceutically acceptablecarrier. By “pharmaceutically acceptable carrier” is meant a carrierthat does not cause an allergic reaction or other untoward effect inpatients to whom it is administered.

Suitable pharmaceutically acceptable carriers include, for example, oneor more of water, saline, phosphate buffered saline, dextrose, glycerol,ethanol and the like, as well as combinations thereof Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the inhibitor.

The pharmaceutical composition or inhibitor may be administered inconjunction with other therapeutic or prophylactic agents. Theseinclude, for example, cyclosporin A, steroids, retinoids, nitrogenmustard, interferon, metbotrexate, antibiotics and antihistamines.

These agents may be administered in single dosage form with theinhibitor (i.e., as part of the same pharmaceutical composition), amultiple dosage form separately from the inhibitor, but concurrently, ora multiple dosage form wherein the two components are administeredseparately but sequentially. Alternatively, the inhibitor and the otheractive agent may be in the form of a single conjugated molecule.Conjugation of the two components may be achieved by standardcross-linking techniques well known in the art. A single molecule mayalso take the form of a recombinant fusion protein. In addition, theinhibitors, or pharmaceutical compositions, useful in the presentinvention may be used in combination with other therapies such as PUVA,chemotherapy and UV light. Such combination therapies may advantageouslyutilize lower dosages of the therapeutic or prophylactic agents.

The inhibitor, or pharmaceutical composition, may be in a variety offorms. These include, for example, solid, semi-solid and liquid dosageforms, such as tablets, pills, powders, liquid solutions, dispersions orsuspensions, liposomes, suppositories, injectable infusible, and topicalpreparations. The preferred form depends on the intended mode ofadministration and therapeutic application. The preferred forms areinjectable or infusible solutions.

The inhibitor or pharmaceutical composition may be administeredintravenously, intramuscularly, subcutaneously, intra-articularly,intrathecally, periostally, intratumorally, intralesionally,perilesionally by infusion, orally, topically or by inhalation.Preferably it is administered subcutaneously, intramuscularly orintravenously. Most preferably, it is administered subcutaneously.

The invention includes formulations suitable for use as topicallyapplied sunscreens or UV-protectants. Preferred embodiments includeLFA3TIP preparations. The active ingredient can be formulated in aliposome. The product can be applied before, during, or after UVexposure, or before, during, or after the development of redness.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly, and are not to be construed as limiting the scope of the inventionin any manner.

EXAMPLES Example 1

Subjects

Six adult patients participated in the investigation. Informed consentwas obtained after Internal Review Board approval of the protocol. Allpatients satisfied the major diagnostic criteria for psoriasis, namelychronic papulosquamous plaques of characteristic morphology anddistribution. -The intermittent use of topical corticosteroids wascommon among these patients but was discontinued 2 weeks prior toentry-into the study. A group of healthy volunteers with no history ofpsoriasis or other skin disease was utilized as the normal controlgroup.

Preparation of Epidermal Cell Suspensions

Skin biopsy specimens were obtained from both normal and lesional skinby using a keratome. The specimens were submerged in Dulbecco'sphosphate buffered saline (“PBS”); (Gibco Labs, Grand Island, N.Y.)containing 50 units/ml dispase (Collaborative Research, Bedford, Mass.).The specimens were then incubated at 4° C. for 18 hours and theepidermis removed from the remaining dermis.

Epidermal sheets were removed from the dermis, submerged in Dulbecco'sPBS containing 0.5% trypsin (Sigma Chemical Co., St. Louis, Mo.), andincubated at 37° C. for 30 minutes.

Trypsinized epidermal sheets were transferred to 0.05% DNase (Sigma) inDulbecco's PBS where they were teased into a cell suspension. Fetalbovine serum (“FBS”) (Hyclone, Logan, Utah) was added to inactivateresidual trypsin and the epidermal cell suspension then passed through a112 μm nylon filter (Tetko, Elmsford, N.Y.). After washing thepredominantly single cell suspension three times in Dulbecco's PBS with1% FBS, cells were resuspended in culture media which consisted of RPMI1640 (Whittaker, Mass.; Bioproducts, Wakerfield, Md.) containing 1%penicillin and streptomycin, 1% glutamine (Gibco), and 10% human ABserum (Sigma).

Preiparation of MNC and T cells

Peripheral blood mononuclear cells (“MNC”) were isolated fromheparinized blood using Ficoll-Hypaque® (Pharmacia) density gradientcentrifugation according to manufacturer's suggested protocol. RestingCD4⁺ T cells were prepared as follows. Macrophages were removed byplastic adherence at 37° C. for 1 hour. The nonadherent,macrophage-depleted MNC ere washed, and then depleted of CD8⁺ Tlymphocytes, activated T cells, B cells, antigen resenting cells and NKcells by incubation with monoclonal antibodies to CD8 (ATCC CRL 8014),HLA-DR (ATCC CRL H355), and CD11b (ATCC CRL 8026). These antibodies wereused as dilutions in PBS (1:200) of ascites fluid from pristane-primedmice.

The antibody treated MNC were incubated at 4° C. with 4.5 nm magneticparticles coated with goat anti-mouse IgG (Dynabeads M-450, Dynal, Oslo,Norway) at a ratio of 3 beads per cell. Antigen positive cells weredepleted by being drawn by a magnet (Advanced Magnetics, Cambridge,Mass.) against the side of the tube allowing the remaining cells insuspension to be decanted. The decanted cell suspension was againexposed to a magnet and cells remaining in suspension collected. Freshgoat anti-mouse IgG beads were again added to the collected cells insuspension in order to deplete any remainmg antigen positive cells, andthe magnetic removal process repeated. Cells were washed in PBS andresuspended in culture media prior to use. This treatment results in apreparation of resting CD4⁺ T lymphocytes enriched to 99% purity anddevoid of intrinsic antigen presenting activity.

Proliferative Response of T Lymphocytes to Autologous Psoriatic Cells

One hundred thousand CD4⁺ T lymphocytes were added to round bottommicrotiter wells (Costar, Cambridge, Mass.) with eighty thousandpsoriatic epidermal cells in 0.2 ml of RPMI containing 10% human ABsenun (Sigmna, St. Louis, Mo.). This number of psoriatic epidermal cellsper well was chosen because previous experiments demonstrated that thisnumber is sufficient to induce autoreactive T cell responses. Afterincubation at 37° C. in 5% CO₂/95% air for 6 days, 1 μCi of [³H]TdR (ICNRadiochemicals, Irvine, Calif.) was added per well and the cellsharvested 18 hours later on a PHD cell harvester (Cambridge TechnologyInc., Cambridge, Mass.). The [³H]TdR incorporation was measured on aPackard scintillation counter (Packard Instrument Co., Downers Grover,Ill.). [³H]TdR incorporation is a measure of T cell proliferation.

Appropriate controls for T cells (“TC”) alone or epidermal cells (“EC”)alone were carried out using the above protocol. No [³H]TdRincorporation was observed in these assays (data not shown). Briskproliferation of autologous T cells in response to psoriatic skin cellswas observed (data not shown).

In addition, to test the allogeneic response to normal skin, the aboveprotocol was carried out using one hundred thousand allogeneic T cellsand eighty thousand normal skin cells. Under these conditions, a briskproliferation of allogeneic T cells was observed (data not shown).

Blocking of Psoriatic Epidermal Cells' Ability to Stimulate Autologous TLymphocyte Proliferation

The effect on [³H]TdR incorporation (i.e., T cell proliferation) of ananti-CD2 monoclonal antibody (TS2/18) (Sanchez-Madrid et al., “ThreeDistinct Antigens Associated with Human T-lymphocyte-mediated Cytolysis:LFA-1, LFA-2,. and LFA-3”, Proc. Natl. Acad. Sci. USA 79, pp.7489-93(1982)), an anti-LFA-3 monoclonal antibody (7A6) (ATCC HB 10695), or anisotype-matched, control monoclonal antibody of irrelevant specificity(MOPC2 1, Sigma Chemical Co., St. Louis, Mo.) was measured using theprotocol outlined above in the presence of 50 μg/ml of the respectiveantibodies.

FIG. 1 demonstrates that addition of anti-CD2 or anti-LFA-3 resulted ina consistent (n=4) and substantial (approximately 60%) inhibition ofautologous T cell proliferation-in response to lesional psoriaticepidermis, as compared to proliferation in the presence of theisotype-matched control antibody.

FIG. 1 displays data for four patients only. These four patientsdemonstrated autoreactivity of blood CD4⁺ T cells to their own lesionalepidermis, despite the fact that no antigen was added to the system.This is an abnormal finding; normal individuals' cocultures ofautologous blood T cells and epidermal cells do not react. Such areaction is considered to be an in vitro model of autoimmune reactionsoccurring in the skin. EC preparations from two additional patients werenot informative. One EC preparation was bacterially contaminated; theother contained antigen presenting cells that did not induceautoreactive T cell responses.

Addition of 50 μg per ml of the anti-CD2 or anti-LFA-3 antibodies to theallogeneic normal skin assay described above also resulted in aninhibition of allogeneic T cell activation. The degree of inhibition wasnot as substantial (approximately 40%) as that observed for autologousantigen presenting cell activity when using lesional psoriatic epidermis(data not shown).

Addition of the isotype-matched control antibody (specific for anirrelevant antigen) did not significantly alter the level of T cellproliferation of autologous T cells induced by lesional psoriaticepidermis (data not shown).

Example 2

Subject

One adult subject participated in this investigation. Informed consentwas obtained after Internal Review Board approval of the protocol. Theminimal dose of UV B from a bank of fluorescent bulbs (FS 40) requiredto induce skin erythema in the subject was determined prior to thestudy. A moderate sunburn (4 minimal erythemal doses) was thenadministered to the left buttock, which 3 days later was the source ofUV damaged skin. Skin from the right buttock, which was unburned, wasutilized for the control.

Preparation of Epidermal Cell Suspensions

Skin biopsy specimens were obtained from both normal and sunburned skinby using a keratome. Epidermal cell suspensions were prepared from thesespecimens using substantially the same protocol as in Example 1.

Isolation and Deletion of T cells

Peripheral blood mononuclear cells (“MNC”) were isolated fromheparinized blood of another person, using Ficoll-Hypaque® (Pharmacia)density gradient centrifugation according to manufacturer's suggestedprotocol. CD4+T lymphocytes were then prepared substantially as outlinedin Example 1.

Proliferative Response of T Lymphocytes to Allogeneic UV DamagedEpidermal Cells

One hundred thousand CD4⁺ T lymphocytes from another individual wereadded to round bottom microtiter wells (Costar, Cambridge, Mass.) withUV damaged epidermal cells from the subject, incubated in the presenceof [³H]TdR, harvested and [³H]TdR incorporation was measuredsubstantially as outlined in Example 1. This example differs fromExample 1 in that the antigenic stimulus is alloantigen, rather thanautoantigens that are stimulatory in psoriasis. Thus, allogeneic T cellswere used, rather than autologous T cells.

FIG. 2 shows a brisk proliferation of allogeneic T cells (as measured by[³H]TdR incorporation) when incubated with UV damaged epidermal cells(“EC+TC”).

Blocking of UV Damaged Epidermal Cells' Ability to Stimulate AllogeneicT Lymphocyte Proliferation

The effect on [³H]TdR incorporation (i.e., T cell proliferation) of ananti-LFA-3 monoclonal antibody (1E6) (ATCC HB 10693), an anti-CD2monoclonal antibody (TS2/18) (Sanchez-Madrid et al., “Three DistinctAntigens Associated With Human T-lymphocyte-Mediated Cytolysis: LFA-1,LFA-2, and LFA-3”, Proc. Natl. Acad. Sci. USA 79, pp. 7489-93 (1982)),and an isotype-matched, control monoclonal antibody of irrelevantspecificity (MOPC21, Sigma Chemical Co.), was measured using theprotocol outlined above in the presence of 50 μg/ml of the respectiveantibodies.

FIG. 2 shows that in the presence of a monoclonal antibody of irrelevantspecificity (MOPC21, Sigma Chemical Co.), [³H]TdR incorporation wassomewhat reduced. However, the addition of anti-LFA-3 monoclonalantibody 1E6 or anti-CD2 monoclonal antibody TS2/18 resulted in asubstantial inhibition of T cell proliferation compared to proliferationin the presence of the control antibody.

Example 3

Preparation of LFA3TIP

LFA3TIP, a fusion protein comprised of the first extracellular domain ofLFA-3 fused to the hinge, C_(H)2 and C_(H)3 regions of human IgG1 wasconstructed as described in Miller, GT et al. (1993) J. Exp.Med.178:211, hereby incorporated by reference. LFA3TIP was purified fromculture medium of transfectant CHO (chinese hamster ovary) cell lines byabsorption to Protein-A Sepharose 4B® (Pharmacia) and eluted with 50 mMglycine, 250 mM NaCl (pH.3.0). Fractions containing protein were pooledand subjected to gel filtration on Superose-6 (Pharmacia) in phosphatebuffered saline (PBS). Peak fractions were pooled and analyzed forpurity on 12% reducing and non-reducing SDS-PAGE.

1. LFA3TIP Results in a Decrease of the Proliferative Response ofAutologous Mononuclear Cells to Lesional Psoriatic Epidermal Cells

The ability of LFA3T1P to inhibit the response of autologous mononuclearcells (MNC) to lesional psoriatic epidermal cells (EC) in suspensionwas-determined. Epidermal cell suspensions were prepared essentially inthe same way as in Example 1 above. Proliferation of MNC was measured bythymidine incorporation. As shown in FIG. 3 there was minimalproliferation with epidermal cells alone. Combination of EC with MNCresulted in a relatively strong response, approaching 6000 cpm. Incontrast to the human IgG controls, addition of LFA3TIP, atconcentrations between 5 and 0.3 μg/ml, resulted in a consistentinhibition of the autoreactive response. If the counts of EC and MNCalone are taken into account, the LFA3TIP inhibition is quitesubstantial.

The mechanism of the IgG1 enhancement of EC-induced MNC proliferation isunknown. It is clear however, from the experiment described above, usingautologous MNC and lesional epidermal cells of psoriasis, that LFA3TIPresults in very substantial inhibition.

2. Spontaneous Proliferation of Lesional Psoriatic Dermal Cells

FIG. 4 shows the effect of LFA3TIP on spontaneous lesional psoriaticdermal cell proliferation. Suspensions of psoriatic dermis were preparedby dispase splitting of the epidermis from the dermis, followed bydigestion of the dermis with collagenase, hyalurinidase, and DNase, andfiltering through sequential nylon mesh sizing filters. Psoriaticdermis, upon digestion into a single cell suspension, undergoes anincreased level of spontaneous proliferation relative to dermal cells insuspensions from normal subjects. The increased proliferation isaccompanied by the formation of clusters, which occurs in psoriatic andnot in normal cultures. The proliferation can only partly be accountedfor by T cell proliferation. There appears additionally to beheterotypic or homotypic adhesion between the dermal cells, some ofwhich may be macrophages and some of which may be stromal cells. Todetermine if LFA3TIP could block at least the T cell component of thespontaneous proliferation, lesional dermal cells were plated insuspension. Approximately 2000 cpm was observed with dermal cells alone(normal controls generally exhibit between 200 and 1500 cpm). A limitingdose of IL-2 was then added at a concentration that should activate onlyT cells expressing the high affinity IL-2 receptor (10 units/ml on adaily basis ×4 days). This boosted the counts to 4600 cpm (dermal cellsplus IL-2). Varying dilutions of LFA3TIP or human IgG were than added. Areduction in spontaneous dermal cell proliferation was seen with doseresponses between 0.03 and 0.003 μg/ml. Furthermore, the LFA3TIPcultures are consistently less proliferative than the human IgG controlcultures through the concentration of 0.01 μg/ml. These results providean interesting and important evidence of LFA3TIP activity on immunologicmechanisms occurring in fresh ex vivo psoriatic tissue.

3. Proliferative Response of Allogeneic Mononuclear Cells to NormalEpidermal Cells

This experiment shows the ability of LFA3TIP to inhibit proliferation ofallogeneic mononuclear cells to normal epidermal cells. Normal epidermalcells were prepared essentially as described for Example 1. MNC wereprepared essentially as described for Example 1. As shown in FIG. 5,epidermal cells (EC) plus MNC demonstrated levels of proliferation ofapproximately 22,000 cpm. Human IgG appears to be slightly enhancingbetween 12 and 0.3 μg/ml of LFA3TIP, relative to EC plus MNC alone.However, LFA3TIP between 5 and 0.1 μg/ml, exerted a cleardose-responsive inhibition. Inhibition of Langerhans cell dependent Tcell activation models the type of activation that occurs in allergiccontact dermatitis, atopic dermatitis and mycosis fungoides type ofcutaneous T cell lymphoma.

4. Proliferative Response of Autologous Mononuclear Cells to LesionalPsoriatic Dermal Cells

FIG. 6 demonstrates the response of autologous MNC to lesional-dermalcells prepared as a single cell suspension. Following dispase splittingof the epidermis from the dermis, the dermis is digested by collagenase,hyalurinidase, and DNase, and filtered through sequential nylon meshsizing filters. In this patient, the spontaneous level of dermal cellproliferation was low, and induction of a dermal cell plus mononuclearcell reaction could be observed. This is a fairly complex system becausethe dermal cell preparation has a number of cell types, includingmacrophages, some neutrophils, antigen presenting cells, filbroblasts,mast cells, and endothelial cells, as well as T lymphocytes. However, itis probably a reasonable approximation of the in vivo milieu as theinfiltrating mononuclear cells first enter into perivascularinterstitial of the dermis in response to chemoattractive signalspresent in psoriasis. Enhancement of the dermal cell-inducedproliferation occurred between 0.3 and 0.01 μg/ml of human IgG control.LFA3TIP inhibited this enhancement, and resulted in a reducedproliferative response between 1 and 0.03 μg/ml. In this dermal cellassay, the addition of IgG or removal of Fc IgG receptor bearing cells,specifically cells bearing the macrophage and neutrophil integrin CD11b,results in elevated spontaneous proliferation of dermal fibroblasts. Towhat degree the LFA3TIP inhibitory effect is upon T cell proliferationas opposed to complex FcγRIU-mediated macrophage antiproliferativeeffects on other cell types is not possible to determine. Regardless,the data clearly support a trend in both this and the previous dermalcell proliferation assays from psoriatic lesional skin which is in thetherapeutically beneficial direction for use of LFA3TIP.

5. Proliferative Response of Allogeneic T lymphocytes to UV DamagedEpidermal Cells

Human subjects were exposed to 4 minimal erythemal doses of ultravioletradiation, and 20 3 days later the epidermis was removed and preparedinto an epidermal cell suspension.

Suspensions were combined with resting, negatively selected CD4⁺ Tlymphocytes which were allogeneic to the sunburned donor. Twoconcentrations of UV-EC are shown in FIG. 7 A and B. Relative to T cellsplus epidermal cells alone, or T cells plus epidermal cells incubatedwith identical concentrations of IgG, LFA3TIP incubation resulted inapproximately 50% inhibition. The cells were moderately free of antigenpresenting cells (APC's), as evidenced by relatively minimal PHAproliferation, but were capable of responding as evidenced byresponsiveness to PMA plus ionomycin. These data demonstrate thatinflammatory UV macrophages which migrate into sunburned skin use anLFA3TIP dependent mechanism to induce T cell activation. Blockade ofthis process should be relevant to photoaging, in that repeatedinflammatory activation in the skin is likely responsible forcollagenase and elastase activation via lymphokine activation. Inaddition, blockade of this signaling may reduce the generation of Tsuppressor cells which these UV induced macrophages generate. Thesesuppressor cells are the one responsible for tolerance in a contactsensitivity mode of UV-induced immunologic host susceptibility to UVcarcinogenesis.

Deposits

Murine hybridoma cells and anti-LFA-3 antibodies useful in the presentinvention are exemplified by cultures deposited under the BudapestTreaty with American Type Culture Collection, on Mar. 5, 1991, andidentified as: Designation ATCC Accession No. 1E6 HB 10693 HC-1B11 HB10694 7A6 HB 10695 8B8 HB 10696

A bacteriophage carrying a plasmid encoding transmembrane LFA-3 wasdeposited under the Budapest Treaty with In Vitro International, Inc.,Linthicum, Md., U.S.A., on May 28, 1987 under Accession No. INI-10133.This deposit was transferred to American Type Culture Collection on Jun.20, 1991 and identified as: Designation ATCC Accession No.λHT16[λgt10/LFA-3] 75107

E. coli transformed with a plasmid encoding PI-linked LFA-3 wasdeposited under the Budapest Treaty with In Vitro International, Inc. onJul. 22, 1988 under Accession No. IVI-10180. This deposit wastransferred to American Type Culture Collection on Jun. 20, 1991 andidentified as: Designation ATCC Accession No. p24 68788Sequences

The following is a summary of the sequences set forth in the SequenceListing: SEQ ID NO: 1 DNA sequence of transmembrane LFA-3 SEQ ID NO: 2Amino acid sequence of transmembrane LFA-3 SEQ ID NO: 3 DNA sequence ofPI-linked LFA-3 SEQ ID NO: 4 Amino acid sequence of PI-linked LFA-3 SEQID NO: 5 DNA sequence of CD2 SEQ ID NO: 6 Amino acid sequence of CD2 SEQID NO: 7 DNA sequence of LFA3TIP SEQ ID NO: 8 Amino acid sequence ofLFA3TIP

While we have hereinbefore described a number of embodiments of thisinvention, it is apparent that our basic embodiments can be altered toprovide other embodiments that utilize the processes of this invention.Therefore, it will be appreciated that the scope of this inventionincludes all alternative embodiments and variations which are defined inthe foregoing specification and by the claims appended hereto; and theinvention is not to be limited by the specific embodiments that havebeen presented herein by way of example.

1-74. (canceled)
 75. An anti-LFA-3 antibody, wherein the antibody is a1E6 antibody, an HC-1B11 antibody, a 7A6 antibody, or an 8B8 antibody.76. The antibody of claim 75, wherein the antibody is a monoclonalantibody.
 77. The antibody of claim 75, wherein the antibody is producedby a hybridoma having an ATCC accession number selected from the groupconsisting of HB10693, HB 10694, HB 10695 and HB
 10696. 78. Ananti-LFA-3 antibody having the sequence of an antibody produced by ahybridoma having an ATCC accession number selected from the groupconsisting of HB 10693, HB10694, HB10695 and HB10696.
 79. A recombinant,chimeric, or humanized antibody derived from the antibody of any one ofclaims 75 to
 78. 80. An antigen-binding portion of the antibody of anyone of claims 75 to
 78. 81. The antigen-binding portion of claim 80,wherein the antigen-binding portion is an Fab fragment, an Fab′fragment, an F(ab′)₂ fragment, an F(v) fragment, a heavy chain monomer,a heavy chain dimer, a light chain monomer, a light chain dimer, or adimer consisting of one heavy chain and one light chain.
 82. Anantigen-binding portion of the antibody of claim
 79. 83. Theantigen-binding portion of claim 82, wherein the antigen-binding portionis an Fab fragment, an Fab′ fragment, an F(ab′)₂ fragment, an F(v)fragment, a heavy chain monomer, a heavy chain dimer, a light chainmonomer, a light chain dimer, or a dimer consisting of one heavy chainand one light chain.